Pathogen-Induced Expression of OsDHODH1 Suggests Positive Regulation of Basal Defense Against Xanthomonas oryzae pv. oryzae in Rice

Reviewer #1

The authors of this paper have provided some editing of the manuscript following the first review. The manuscript, however, requires further editing and/or repeat of experimental work prior to publication.

Authors would like to thank the Reviewer for his time and constructive comments and suggestions to improve our manuscript. Most of the comments have been addressed, including those highlighted in the PDF version of the manuscript by the Reviewer.

The major concern is the lack of clear understanding about bacteria inoculum concentrations.

It appears the study was done with a very weak inoculum, significantly lower than normally used by plant bacteriologists for pathogenicity testing. The references provided in the manuscript further support this as those studies used inoculum in the order of 108 – 109 CFU/ml whereas this current study as only used 0.002 CFU/ml.

1.        

We apologize for the inconvenience. We used a concentration of xx × 10^xx CFU. mL^-1 in our experiment A detail explanation is also given in response to the next comment.

The google patent reference should be removed as it provides no detail on methodology. This discrepancy with inoculum concentration brings doubt to the results presented, particularly as the study is focused on plant response to bacterial challenge.

This is poor methodology and the reference provided is not appropriate. It is not possible to confirm this bacterial concentration is appropriate from the google patent reference provided. The CFU/ml reported is very unusual compared to most other plant bacteriology studies.

The reason to do dilution platings and count colonies is to confirm the viable CFU/ml used as the OD is only an estimate of cells present, a proportion of which will no longer be viable. This makes interpretation of the transcript response difficult when there is no real measure of much bacteria the plants were challenged with. The disease symptoms are helpful but having a more quantitative measure of the expose levels would significantly strengthen this paper (line 167)

The authors appreciate the concern raised by the worthy reviewer. We apologize for the inconvenience regarding the reporting of the inoculum concentration. We have described the bacterial growth method and have clarified as follows:

Lines 182-187 of the revised manuscript:: The actual concentration of the bacterial suspension culture had an OD₆₀₀ equal to 1.573 (equivalent to 1.3 10⁹ CFU mL^-1) was recorded using a Spectrophotometer (AA6300C, Shimadzu, Japan). For inoculation to rice plants, this concentration was adjusted through serial dilutions to the absorbance (OD₆₀₀) of 0.002, which corresponds to about 1.6 10⁶ colony forming units (CFU mL^-1). However, we have mistakenly written the OD value rather than orignal concentration. We have revised this in the manuscript now. This concentration has been used by (paper reference). We have now cited this original paper in the concerned methodlogy sectoin instead of the patent.

Furthermore, the section on Arabidopsis should not be included in the manuscript as there is insufficient results provided and the results do not clarify the role of the gene under study.

This is not strong enough reason to include this work in this manuscript. Also the results presented does not support the authors hypothesis as there was very little response of the gen in Arabidopsis

We appreciate the concerns raised by the reviewer, and the interest to improve the manuscript. However, we suggest that removing Arabidopsis data will disrupt our story line and weaken our hypothesis.

Owing to the difficulties in getting rice mutant line for OsDHODH1, which is explained in the introduction section of the revised manuscript line 94-102, we used its homolog (AtPYD1) in Arabidopsis which is another widely used model plant. We agree to the point that Arabidopsis work could be published separately after adding some experiments, but we would like to include it here to support our argument. It further suggests that OsDHODH1 function might be conserved both in monocots and dicots.

Therefore, in the introduction section, lines 94–102, we have included the following:

AtPYD1 gene was upregulated in Col-0 about 2-folds following inoculation of Pst DC3000 virulent bacteria. Furthermore, this increase in the expression of AtPYD1 was statistically equivalent to the increase in th expression of pathogenesis-related genes (PR genes), which are well-established marker genes for pathogen response in plants. Besides, the phenotypic response indicated that the Arabidopsis knockout atpyd1-2 plants exhibited a high susceptibility towards virulent strain of the bacterial pathogen Pst DC3000 as compared to the Wild type Col-0 plants. Similarly, the atsid2 and atgsnor1-3, used as susceptible controls showed similar phenotypic response when compared to atpyd1-2. This suggests a key role for the AtPYD1; which is an orthologue of OsDHODH1 in the regulation of plant defense responses to bacterial infection.

Further work is needed on the Arabidopsis gene before publication and can be presented as a separate publication.

2.        

We sincerely appreciate the concern raised by the reviewer, and the interest to improve the manuscript. We would like to specify that this study was limited to the basal defense level to investigate the transcriptional regulation of the target genes compared to the PR genes and the phenotypic response in support to the rice experiment. We take note of the good suggestion, and future studies that may include Systemic Acquired Resistance (SAR) and R gene-mediated defense and all related experiments may be conducted for another independent publication.

More detail is needed in some methods sections (see manuscript)

General comments about the manuscript:

The manuscript requires further editorial work as there are spelling mistakes and inconsistencies with formatting of units etc. Some examples are shown on the manuscript.

The order of the sections presented needs reconsideration. Normally pathogenicity sections are presented first as these results of these experiments are used subsequently in other areas of work such as the gene marker evaluation and then host response work.

The resistance gene marker section also requires further clarification and rewriting. Specific comments are on the manuscript.

We appreciate the comments made by the reviewer. Therefore, we would like to indicate that the we have improved the materials and method, results section, and the discussion. We also checked for typo or grammatical errors throughout the manuscript.

A space is required between the number and the unit -1 h not 1h. The exception is for a percentage where 1% is correct. Please review the manuscript and edit as needed.

We appreciate the Reviewer’s interest to improve the manuscript. All the necessary changes have be made throughout the manuscript as suggested.

Further expansion of the results from this work is needed. There is only a brief mention in the results to say the amplicon size was confirmed by sequencing. The marker analyses in general needs to be reconsidered and rewritten.

We are thankful to the Reviewer for his constructive suggestions. We have included description about the cloning and sequencing of Xa21 gene (Lines 155-159 and 162-175).

Incorrect spelling – cycloheximide (line 163)

Line 180: we have included the correct spelling as suggested.

What does the T-DNA insertion refer to? What is the method used for the PCR?

Detailed method of PCR is given for the marker work but not here? This is inconsistent presentation of methodology.

The primers used should be listed in the manuscript not in a supplement table and the PCR conditions provided.

The T-DNA (transfer DNA construct) insertion is referring to the DNA of the tumor-inducing (Ti) plasmid of some species of bacteria, such as Agrobacterium tumefaciens transferred into the host plant’s nuclear DNA genome through the process of transformation to induce mutations.

Since the PCR conditions for genotyping using DNA markers and the one for genotyping to detect T-DNA insertion lines are similar, with an exception on the annealing temperatures and the type of primers, which is selected based on the primers information, we found no reason to duplicate this section to avoid inflating the manuscript. Therefore, we have modified the subsection 2.7. as follows (Lines 210-216):

“The atpyd1-2 plants were genotyped to identify homozygous T-DNA (transfer DNA) insertion lines by polymerase chain reaction (PCR) for further experiments (Figure S4). The T-DNA insertion lines were identified through genotyping, using left border (LB) and gene specific reverse primers, and the DNA samples extracted from the atpyd1-2 plants, following the DNA extract method and PCR conditions described earlier in 2.3. subsection.”

The list of primers have been moved to the main text, now referred to as Table 1 (Line 122)

In this real-time PCR or RT-PCR which refers to reverse transcriptase PCR (i.e. detecting RNA targets not DNA target). Please be clear

We thank the Reviewer for the concern. We would like to specify that we were referring to the genotyping by polymerase chain reaction (PCR) to identify homozygous lines, using primers specifically designed for the detection of the T-DNA insertion. Therefore, we have made necessary changes in the manuscript as follows (lines 210-216):

«The atpyd1-2 plants were genotyped to identify homozygous T-DNA (transfer DNA) insertion lines by polymerase chain reaction (PCR) for further experiments (Figure S4). The T-DNA insertion lines were identified through genotyping, using left border (LB) and gene specific reverse primers, and the DNA samples extracted from the atpyd1-2 plants, following the DNA extract method and PCR conditions described earlier in 2.3. subsection. Primers for genotyping were designed using the SALK_083897C (the Arabidopsis identification number of the target mutant line) in the iSect primer tool found in the following link: http://signal.salk.edu/tdnaprimers.2.html »

Inconsistency in the use of units – h or hours not both (line 201)

We have harmonized the use of units throughout the manuscript as suggested

Typo assume this is 10^-5 – also inconsistency with units choose CFU/ml or CFU ml^-1 and use throughout the document

We apologize for the inconvenience. We have made the necessary changes accordingly.

Please use only one acronym – if you say quantitative there is no need to include real-time as well as its implied (Line 227)

We appreciate the suggestion made by the Reviewer, and we have made the necessary changes accordingly (Lines 247-248)

Further detail is needed on the housekeeping genes – which primers, etc. The primers for the OsDHODH1 and the resistance genes should also be presented here not in the supplement (line 234)

We would like to indicate that all information regarding the genes and the sequences of primers used in the study are organized in the Table 1 (previously Table S1). We found this way more convenient (Line 122)

This results section is very detailed, however, the interpretation of the results is unclear. I assume that marker assays were done to evaluate if the rice cultivars contained resistance genes and the presence/absence of these genes is indicated by the size of the PCR amplicon.

Given that it appears none of the PCR assays gave positive result for the resistance genes the description of the results could be simplified to say just that. There is also no need to present Figure 1.

If this is not correct and a smaller or different sized band is indicative of presence of a resistance gene the…

We would like thank the Reviewer for his constructive comments to improve our manuscript. Therefore, we would like specify that the polymorphism between lines and the bigger band size of the Xa21 and xa5 is indicative of the presence of the resistance genes in the detected rice cultivars. We also indicated that previous have shown different banding sizes of the Xa21 using the same STS DNA marker. Some reported 1018 bp other about 950 bp, and in our case, we reported 733 bp indicating the presence of the resistance gene for Xa21. We can also see in the panel a, b, c, and d that the markers used detected resistance alleles of the Xa2, Xa4, xa5 and Xa21, respectively.

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Replace …at early time of … with ..soon after (line 281_

We have replaced at early time of …with soon after bacterial infection as suggested (line 346)

Delete ..which showed resistance, and moderately susceptible (interspecific, Nerica-L14).

We have deleted as suggested

Results presented here don’t show downregulation as there is no negative values, results just show a low response in the susceptible cultivar compared to the resistance one

We appreciate the concern raised by the reviewer. However, would like to specify that the results indicated downregulation of the expression of the studied genes, when you compared to the basal level. These results are showing the relative expression of the target genes in both controls and Xoo inoculated plants. The negative values appear when one calculates the logarithm base 2 (log2) of the fold change (FC is obtained by dividing the relative expression in treated samples by that of the control). But the meaning remains the same.

Line 292: add K3

The paper would read better if re-ordered to present the pathogencity data first. The transcript analyses is done on cultivars chosen from these results thus they should be presented first. It also makes the marker results easier as the reader knows which cultivars were susceptible etc. before reading these later sections (Line 292)

We are thankful to the Reviewer’s for his constructive suggestion. Therefore, we have reorganized the results section, and the 3.3 is now 3.2 after the genotyping results (Lines 295-307).

Line 295: inconsistency - assume this should be CFU/ml not OD according to the methods section?

We have corrected the report of the bacterial density, and have indicated the values in CFU mL^-1) throughout the manuscript

Line 304 : delete ...all cultivars put together

We have delete from the manuscript as suggested

Figure 3 and Table 2 present the same data, there is no need for both

We have delete previous Figure 3 from the manuscript, but we have kept Table 2 (now Table 4)

Line 320: add… to Xoo-K3 inoculation

We have specified the host response to to Xoo-K3 inoculation as suggested (Table 4, line 308)

Line 333: This reference provides no easily found information such as evaluating BLB by time

We have provided the full link (line 321)

The variability in pathogenicity observed is most likely a result of using an incorrect concentration of inoculum to challenge plants (Line 339)

3.        

Authors reiterate their observation, considering that the issue regarding the bacterial concentration has been resolved with the clarifications given earlier.

The results presented in this section do not support the original hypothesis of the authors. There is inefficient investigation of this gene to warrant inclusion in this manuscript. It is very unclear how the plant is responding with this limited experiment. Further work is needed to better understand the responses and then publication as a separate study at a later date.

Authors appreciate the point raised by the Reviewer. However, we reiterate the consideration to maintain the Arabidopsis data, and have considered that enough explanations have been given earlier. Otherwise, the substance of the study will be diluted.